F. F. Johansen

Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat

The fluorescent dye 6-methoxy-8-p-toluene sulfonamide quinoline (TSQ) was used to monitor the distribution of zinc in the hippocampus and fascia dentata of adult rats subjected to 20 min of cerebral ischemia. In normal brains TSQ stains only neuropil, in particular the mossy fiber layers in the dentate hilus (CA4) and CA3, but within 2 h after ischemia, TSQ-fluorescent cells were observed in the dentate hilus. At longer survival times TSQ-positive cells stained positively with acid fuchsin, a sign of cellular degeneration. At the same time a decrease in the TSQ fluorescence of the mossy fiber terminals in the dentate hilus (CA4) and the CA3 mossy fiber layer was noted. The observations suggest that zinc many play a role in the selective death of dentate hilar neurons after cerebral ischemia.

Early loss of somatostatin neurons in dentate hilus after cerebral ischemia in the rat precedes CA-1 pyramidal cell loss.

SummarySomatostatin (SS)- and cholecystokinin (CCK)-immunopositive cell somata in the rat hippocampus were quantitated at day 1, 2, 3 and 4 after cerebral ischemia. A significant (P<0.01) 60%–80% loss of hilar and CA-3c SS neurons took place. No CCK neurons were lost. Damage to SS neurons was significant on the second postischemic day and preceded the delayed loss of CA-1 neurons. We speculate that loss of SS neurons, which presumably innervate the inhibitory GABAergic (γ-aminobutyric acid) interneurons, may induce hyperactivity stimulating the Ca-1 neurons to death.

Resistance of hippocampal CA-1 interneurons to 20 min of transient cerebral ischemia in the rat

SummaryThe aim of this morphological study was to determine the vulnerability of hippocampal interneurons to ischemia in the adult rat. Two types of interneurons situated in the CA-1 stratum oriens were investigated, the larger basket cells close to stratum pyramidale and the smaller basket cells close to the alveus.Male Wistar rats were subjected to 20 min of transient cerebral ischemia by means of 4-vessel occlusion and perfusion fixed 1, 2, 4, or 21 days later. In both Golgi-impregnated and in routinely stained sections the pyramidal cells and interneurons in the hippocampal CA-1 region were examined and counted.The study clearly demonstrated the selective vulnerability of the CA-1 pyramidal cells, as no ischemic cell damage to or loss of interneurons was found.

Removal of the entorhinal cortex protects hippocampal CA-1 neurons from ischemic damage

SummaryThe excitatory (glutamatergic) innervation seems to determine a nerve cells vulnerability to complete, transient ischemia. Interruption of the excitatory afferents to the hippocampus by removal of the entorhinal cortex prior to ischemia allows examination of this hypothesis. Groups of adult male Wistar rats were subjected to 20 min of ischemia (fourvessel occlusion) 4 days following a sham procedure, unilateral or bilateral entorhinotomy. CA-1 pyramidal cell survival following ischemia was assessed by light microscopic examination (cell counts) 4 days after ischemia. Compared to control animals unilateral entorhinotomy protected 50% of the CA-1 pyramidal neurons ipsilateral to the lesion, whereas bilateral entorhinotomy resulted in 84% protection. The pathophysiology of ischemic brain damage is discussed, and it is suggested that the protection of CA-1 pyramidal neurons after entorhinotomy is due to interruption of the input to the dentate granule cells, which forms a link in the trisynaptic pathway from the entorhinal cortex to the CA-1.

Ischemic CA-1 pyramidal cell loss is prevented by preischemic colchicine destruction of dentate gyrus granule cells.

Hippocampal CA-1 pyramidal cell damage was produced by 20 min of cerebral ischemia. Colchicine destruction of the dentate gyrus granule cells 11 days before ischemia prevented the CA-1 cell loss. It is suggested that the protective effect of degranulation on ischemic CA-1 pyramidal cell damage is due to reduction of glutamate release in CA-1 during and after ischemia.

Protection against ischemic hippocampal CAI damage in the rat with a new non-NMDA antagonist, NBQX

Two glutamate antagonists were tested in a rat model of complete, transient cerebral ischemia. Six days after 10 min ischemia the mean loss of hippocampal CAI pyramidal neurones was 73%. Administration of the AMPA (a‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole proprionic acid) antagonist NBQX (2,3‐dihydro‐6‐nitro‐7‐sulfamoyl‐benzo(F)quinoxaline) reduced the pyramidal neurone loss to 1%, 11% and 15%, when given before, immediately after or 1 h after ischemia, respectively. MK‐801 (dizocilpine), a competitive NMDA antagonist gave no protection in this model. We suggest that the AMPA receptor transduction mechanisms are sensitized by ischemia and that the postischemic blockade of the main glutamatergic input to the CA 1 cells with NBQX impairs the deleterious effect of “normal” postischemic excitatory transmission.

Neural grafting to ischemic lesions of the adult rat hippocampus

SummaryThe purpose of this study was to examine the structural and connective integration of developing hippocampal neurons grafted to ischemic lesions of the adult rat hippocampus. The 4-vessel occlusion model was used to cause transient cerebral ischemia which damages CA1 pyramidal cells in the dorsal hippocampus, but spares nonpyramidal neurons and afferents in the area. One week later, cell suspensions were made from the CA1 region of fetal (E18-20) rats and injected stereotaxically into the lesion. The recipient brains were examined 6 weeks to 6 months later for survival, morphology, and intrinsic and extrinsic connections of the grafts. The methods used included cell stains, histochemical staining for acetylcholinesterease (AChE), immunocytochemical staining for neuropeptides (cholelecystokinin (CCK), somatostatin (SS), enkephalin (Enk) and an astrocytic marker, glial fibrillary acidic protein (GFAP), as well as tracing by retrograde axonal transport of fluorochromes and light and electron microscopy of anterograde axonal degeneration. The grafts survived well (80%) and were often quite large. They were well integrated in the lesioned host brain area, contained both pyramidal cells and neuropeptidergic neurons and displayed a near normal GFAP immunoreactivity for astrocytes. The latter contrasted the dense gliosis of the host ischemic lesion. Judged by the AChE staining the grafts were innervated by cholinergic host septohippocampal fibers. Ingrowth of host hippocampal commissural fibers was demonstrated by Fink-Heimer staining for degenerating nerve terminals following acute lesions of the hippocampal commissures. At the ultrastructural level degenerating, electron dense terminals of host commissural origin were found even deep inside the graft neuropil in synaptic contact with mainly dendritic spines. A transplant efferent connection to the host brain was demonstrated by retrograde fluorochrome tracing and consisted of a homotypic projection to more posterior levels of the ipsilateral host CA1 and subiculum. Minor abnormal, efferent projections to the host dentate molecular layer were shown in Timm staining. We conclude that fetal CA1 neurons grafted to one week old ischemic lesions of the dorsal CA1 in adult rats become structurally well incorporated and can establish nerve connections with the host brain.

Increase in neurogenesis and behavioural benefit after chronic fluoxetine treatment in Wistar rats.

Objectiveu2002–u2002 Disturbances in hippocampal neurogenesis may be involved in the pathophysiology of depression and it has been argued that an increase in the generation of new nerve cells in the hippocampus is involved in the mechanism of action of antidepressants.

Chapter 7 Glutamate receptor transmission and ischemic nerve cell damage:evidence for involvement of excitotoxic mechanisms

Publisher Summary Cerebral ischemia has been studied in a number of animal models, mainly with rats. Although the most popular ones differ somewhat with respect to the neuropathological changes, often a hierarchy within the vulnerable cells can be identified. The development of morphological changes (acidophilia, eosinophilia) takes from a few hours to four days or longer. More severe ischemia results in an earlier occurring eosinophilia and vice versa, the so-called maturation phenomenon. In studying the influence of the excitatory (glutamatergic) input on the ischemic neuronal damage, the use of short ischemia is necessary. Neuropathological examination some days after an episode of cerebral ischemia reveals that a number of cell types are involved. The hematoxylin–eosin stain or acid–fuchsin stain are standard for the detection of eosinophilic neurons, but the Fink-Heimer impregnation is more suitable to show both dead nerve cell bodies as well as their axons and dendrites, and is the superior method if the distribution of ischemic damage must be mapped.

Post-ischemic and kainic acid-induced c-fos protein expression in the rat hippocampus

ABSTRACT The c‐fos protein is a gene regulatory third messenger involved in long‐term responses of cells to various stimuli. It can be used as a marker of neuronal activity. In the present immunohistochemical study the presence of c‐fos protein (FP) in the rat brain from 1 h to 14 days after 10 min of cerebral ischemia was compared with that 3 h after an intraventricular injection of kainic acid. The kainic acid injection resulted in staining of dentate hilar cells, granule cells and hippocampal interneurones. The postischemic changes at Day 1 were sporadic CA1 pyramidal cells expressing the FP. At Day 2 FP was expressed with variable intensity in many pyramidal cells in the CA1. At Day 3 many necrotic CA1 pyramidal cells were seen. They did not express the FP, and the expression was less intense and found in fewer cells than at Day 2. At Days 3, 7 and 14 there was increasing gliosis without c‐fos expression in the CA1. The study demonstrates a delayed postischemic synthesis of the gene regulatory protein c‐fos preceding the necrosis in the selectively vulnerable CA1 region.